The Tox21 program is a federal collaboration among researchers from NIH, including NCATS and the National Toxicology Program (NTP) at the National Institute of Environmental Health Sciences (NIEHS), the Environmental Protection Agency (EPA), and the Food and Drug Administration (FDA).. These agencies work together to advance in vitro toxicological testing. The Tox21 Program is comprised of the following specialties: Systems Toxicology, Genomic Toxicology and Computational Toxicology. The Systems Toxicology team has identified, developed, optimized and/or screened more than 18 assays. Highlights include performing 10 online screens, including real-time cell toxicity assays in Hek 293 and HepG2 cells, pH2AX assay, hypoxia assay and constitutive androstane receptor (CAR) assay, in triplicate against the Tox21 10K compound collection and eight online validation assays, including G-protein coupled receptor (TSHR), signaling pathway and nuclear receptor assays, against the LOPAC collection on the Tox21 robotic system. All of these assays were optimized and evaluated before moving to robotic online validation and online screening. Tox21 program researchers completed the Tox21 Data Challenge 2014, a crowdsourcing competition to develop computational models based on the 10K data generated from 12 assays that were tested by the Systems Toxicology team. These assays are estrogen receptor alpha (ERa) ligand binding domain (LBD), estrogen receptor alpha (ER, full length), aromatase, androgen receptor (AR, full length), androgen receptor LBD (AR, LBD), aryl hydrocarbon receptor (AhR, full length), peroxisome proliferator-activated receptor gamma (PPAR-gamma), nuclear factor (erythroid-derived 2)-like 2/antioxidant responsive element (Nrf2/ARE), heat shock factor response element (HSE), ATAD5, mitochondrial membrane potential (MMP), and p53. In collaboration with the Hamner Institutes for Health Sciences and the NTP, the Systems Toxicology team has tested 60 fruits and vegetable extracts in a 384-well plate format in the CAR assay in agonist, antagonist and viability modes. The team also has screened the Tox21 10K collection against the DNA-repair-deficient clones and selected 15 compounds based on the differential activity responses. Several follow-up studies have been performed (e.g., micronuclei and pH2AX assays), and about 150 cherry-picked compounds have been tested. Three compounds, 2-oxiranemethanamine, AD-67, and tetraphenylolethane glycidyl ether, with previously undefined genotoxicity were identified as genotoxic. The manuscript with these findings has been published in Mutagenesis this year. As a follow-up study for the FXR project, approximately 200 potent and reproducible FXR-active compounds identified from the Tox21 10K screen have been further tested in FXR-bla assay and CMV-bla control counterscreen on FXR-active Tox21 chemicals to identify false positive compounds as results of beta-lactamase inhibition, transcription inhibition, or other nonspecific effects. In addition, the team has worked on the mitochondria project and selected about 800 compounds to be tested in rat hepatocyte MMP, reactive oxygen species (ROS), ATP content and caspase 3/7 assays. In collaboration with NIEHS, a nuclear receptor redistribution assay has been optimized and 3 DDT-related contaminants and metabolites have been tested for their ability to disrupt redistribution of the estrogen receptor alpha in U2OS human bone osteosarcoma cells. The team also has searched for epigenetic assays for the Tox21 Program by optimizing a cell-based HDAC I/II assay in various cancer cell and stem cell backgrounds and validating this assay by screening the NCATS Pharmaceutical Collection of 2,816 approved and investigational drugs. A manuscript is under preparation for this investigational study. The TSHR project is a collaborative effort with NCATS and the EPA to generate and evaluate the TSHR cell line by measuring cAMP levels after TSH stimulation. The Androgen (AR) project analyzes the AR-agonists and antagonists identified from the Tox21 10K screening. The manuscript for this project is in preparation and will report on several important structural classes with a focus on confirming the large screen results with a biochemical assay determining the binding affinity of selected compounds to the AR-LBD. In addition, the team has evaluated estrogen-related receptor alpha (ERRalpha)/proliferator-activated-receptor-gamma coactivator-1alpha (ERRa/PGCa) and ERRa cell lines by luciferase reporter gene expression. This year, NCATS initiated a development biology project funded through NCS. In collaboration with the Johns Hopkins University, Xias group started to profile the effect of the Tox21 chemicals on the neural stem cells derived from normal, autistic and schizophrenic patients.